The present invention relates to novel phenylalanine ammonia-lyase-producing microorganisms and methods for their selection, production, and use. More particularly, the invention concerns microorganisms which produce relatively high levels of the enzyme, phenylalanine ammonia-lyase (hereinafter sometimes called PAL), which in turn, is useful for the production of L-phenylalanine.
L-phenylalanine is an essential amino acid in man, and is, therefore, an important ingredient of enteral and parenteral nutritional formulations. In addition, this amino acid is useful as a starting material for the production of other products, such as the artificial sweetener, aspartame. Various microbial processes for the production of phenylalanine are known. For example, U.S. Pat. No. 3,660,235 describes the production of phenylalanine by phenylalanine analog resistant strains of Brevibacterium, Corynebacterium, Arthrobacter, Bacillus and Candida. The production of this amino acid by tyrosine-requiring mutants of certain strains of Brevibacterium, Corynebacterium, Arthrobacter, and Escherichia is also known. See U.S. Pat. Nos. 3,654,079 and 3,909,353.
PAL catalyzes the breakdown of L-phenylalanine to trans-cinnamic acid and ammonia. This enzymatic reaction is reversible, and British Patent No. 1,489,468 discloses a process for the production of L-phenylalanine which involves the PAL-catalyzed reaction of trans-cinnamic acid with ammonium ions to yield L-phenylalanine. This reaction has been found to be a useful procedure for producing L-phenylalanine, and therefore, there is a continuing need to obtain production microorganisms which produce high levels of PAL activity. Such microorganisms can be used directly for the conversion of cinnamic acid and ammonium ions to L-phenylalanine, or the enzyme can be isolated from the cells and used to produce L-phenylalanine in various forms of bioreactors.
One problem that has been associated with the microbial production of PAL is that its biosynthesis is repressed by carbon catabolites. The induction of PAL biosynthesis therefore requires media that are substantially free of glucose or other simple carbohydrate nutrients. Since the growth media used for cultivating microbial cells to a satisfactory cell density advantageously contain glucose or other carbohydrates, it has heretofore been necessary to remove those nutrients prior to PAL induction. Such removal has generally been accomplished by permitting the cells to consume substantially all available carbohydrate nutrients prior to induction. An alternative procedure has been to separate the cells from their growth medium and resuspend them in a catabolite-free induction medium. PAL production could be simplified and facilitated by using PAL-producing cells that are resistant to catabolite repression.